For some time, the art has known that one can clone a foreign gene of interest, and then express it in a suitable host to produce a desired proteinaceous material. Typically, this has been done by inserting a foreign gene (or cDNA version thereof) into a vector (e.g., a plasmid, a virus), and then transforming the host (e.g., a bacteria) with the vector. Commercial production processes for proteins of interest have been built around these concepts.
To improve efficiency and reduce costs, the art has looked for ways to have multiple copies of the foreign gene of interest in a single cell ("amplification"). One commonly employed amplification procedure involves insertion of the foreign gene into a "high copy number" type plasmid, with the subsequent introduction of the plasmid into a suitable host. The host then reproduces a high number of copies of the plasmid inside the cell, thereby creating multiple copies of the gene to be expressed.
However, plasmid instability (and thus a low yield) may result if the inserted genes adversely influence plasmid replication or maintenance.
Thus, the art sought to develop a biological system that automatically produces tandem repetitions of a foreign DNA sequence in the chromosome of a host. In J. Altenbuchner, et al., 201 Mol. Gen. Genet. 192-197 (1985) (the disclosure of this article and all other articles recited herein are incorporated by reference as if fully set forth herein) part of a Streptomyces lividans gene sequence which underwent internal amplification was identified. A foreign gene was then inserted in the sequence and amplification of the foreign gene resulted. However, this system probably resulted in mixtures of amplified strains, and is complex.
Thus, a need has existed for an improved means of amplifying foreign genes to be used in protein production independent of the continued presence of a plasmid.